Nextera mate pair library preparation protocol

The DNA sample was sent to Laboratório Central de Tecnologias de Alto Desempenho em Ciências da Vida (LaCTAD, UNICAMP, Campinas, Brazil) for quality check (2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA), library preparation, and sequencing. Library preparation was done using Illumina Nextera kits (Illumina, San Diego, CA), and paired-end and mate pair sequencing was done on a HiSeq 2500 platform (Illumina). The extracted DNA was used for the construction of three sequencing libraries: two paired-end (one lane each) and one mate-pair (one lane). The paired-end libraries were prepared according to the TruSeq™ DNA Nano Library Preparation Protocol (Illumina) using 100 ng input DNA. After DNA shearing, 350 bp inserts were selected using a bead-based method. Inserts were amplified by 8 PCR cycles, and the sequencing reaction yielded 2 × 101 bp reads. The mate-pair library was prepared from 1 μg of input DNA, following the Nextera® Mate Pair Library Preparation Protocol (Illumina). Fragments of 3 kb were circularized and sheared followed by purification of mate-pair fragments using beads. Mate-pair fragments were amplified in 10 PCR cycles, and the sequencing reaction produced 2 × 101 bp reads.

Genomic DNA was extracted from liver tissue of an adult animal following a standard phenol-chloroform extraction protocol. MiSeq libraries were prepared following a standard Illumina DNA library preparation, which involved shearing of DNA to 550 bp by sonication (Covaris S2), XP bead purification (Beckman Coulter), end polishing, A-tailing, and ligation of indexed adapters (TruSeq DNA PCR-Free kit, Illumina). After ligation, adapters were depleted by XP bead purification (Beckman Coulter). Libraries were quantified by qPCR with the KAPA library quantification kit (KAPA Biosystems), and equimolar pooled for sequencing. To generate mate-pair libraries, purified DNA was subjected to the Nextera Mate Pair Library Preparation protocol (Illumina). For accurate sizing after tagmentation, gel-based size selections for an average of 2 Kb and 10 Kb were done. The resulting libraries were quantified with Fragment Analyzer (Advanced Analytical) and equimolar pooled for sequencing. We sequenced 2 × 300 bp reads from three fragment libraries on the Illumina MiSeq platform, and 2 × 150 bp and 2 × 100 bp reads from two 2 Kb and two 10 Kb mate-pair libraries on the Illumina HiSeq 2500 platform to a total sequencing coverage of 104X (Supplementary Table 1).